It is known that Zebra fish show color preference for both red and blue when placed against other colors such as green and yellow (Park, 2016), but little is known about the development of color preference in tadpoles such as Xneopus laevis.  Because of this, myself, Joseph Owens, and Jillian Dabbraccio decided to look further into the color preference of Xneopus laevis with red and blue stimuli using an I maze.  While no statistically significant data was found, we do believe that with further testing with different resources would provide valuable and possibly significant results.

Video 1.  Average vs Outlier.  This video is of two trials performed sped up 5x (two one minute clips) with the average tadpole first and the outlier second.

Methods

To perform this experiment, 12 tadpoles were chosen at random and deprived of food for 24 hours and kept in 10% pond water.  An I maze was constructed (30 cm x 8 cm x 3.3 cm) of non-transparent white plastic, which had small notches of plastic removed for the insertion and removal of a variety of other plastic pieces.  These notches were spaced 1 cm from the back wall, the edge of the next wall was 5 cm from this wall, and 5 cm separated this wall from the center notches.  The center notches were located 2.5 cm apart and served as the acclimation center for the tadpoles.  Individual tadpoles were placed into the acclimation center for 3 minutes before the test began, with the same white non transparent plastic material panels acting as boundaries to contain the tadpole before the experiment began.  Each trial used this and one red panel and one blue panel (which swapped sides each test) located 1 cm from each end of the I maze, as well as 150 mL of 10% pond water which was changed for each individual, and .01 g of food behind the red and blue panels.  Using the edge of the second notch closest to the ends of the maze, we determined the tadpoles were in the red or blue zone.  After the 3 minute acllimation period, the center panels were removed allowing the tadpole to freely roam the I maze.  This period lasted 5 minutes and each change in location (blue to middle/red to middle/ middle to blue/red) was marked by the new location and the time the tadpole entered the location.

Figure 1. I Maze. Above is the I maze used in the Xenopus laevis color preference trials.  The acclimation center, as well as the blue and red zones are marked to show where the area in which the results were measured.  Note: food used were not actual steaks.

Results

Below are a graph and figure that depict the data collected in the experiment. The graph shows the time spent in the red or blue side of the maze on the vertical axis and the individual tadpoles on the horizontal axis. The figure below showcases our data from one of the average tadpoles in order to illustrate how we recorded our data during the experiment. A t-test was used to look for significance in the mean times spent on each side (red=66.92 sec.) (blue=87.17 sec.) which gave a P value of 0.5294.  This showed that there was no statistically significant difference in data between the time spent in each of the respective colored areas.

 

Figure 2. Time spent in each color area. This graph shows the time (in seconds) each individual tadpole spent in both the red and blue areas.

 

 

 

 

 

Table 1. Tadpole 12 trial.  This table shows how data was measured for an average tadpole in our experiment, including the time the trial was run, the location of the red and blue panels, and the sum of time spent on each side in seconds.

 

 

 

 

Discussion

While the data collected does not show a statistically significant difference in time spent in the red area compared to the blue area, I think there is an abundant amount of room for improvement in the how this experiment was conducted.  The most important change that would need to be made if this experiment were run again is the I maze itself.  Figure 3 shows the maze used in the experiment performed with zebra fish to observe any of their color preference.  Each segment of their “+” maze has the sides completely covered in the color that was being tested for.  This eliminates the possibility of having a lack data seen in our experiment due to the tadpole primarily remaining in the middle of the maze where nothing was measured (see 2nd trial in video 1).

Figure 3. Maze from (Park,2016).  Mazes have measured sections completely covered eliminating the chance of a tadpole being in an uncolored area.

While I believe figure 3 shows an example of a maze that would give better results than what we ran, I believe this could also be improved.  There are a number of ways this could be done, such as coloring the floors with the same colors as well.  While this might not have been an issue with the use of the zebra fish, tadpoles spend a considerable amount of time with their tales up and heads facing the ground, something we even observed in our trials, but this does not completely solve every issue.  This distance of how far the light from each panel can travel will vary depending on how opac the plastic being used is.  This was another issue we ran into, but is another variable that must be kept in thought when trying to show preference with absolutley no other variables.  I would argue using colored lights, of the same wattage, and same distance away could be the best method to “cover” an area in color.  This would leave no area that the amount of color recieved would be more or less than another area, which could be argued in maze shown in figure 3.  Other variables that could have improved the results seen in our experiment would be the movement of the tadpoles which may have been slower due to the time period without food, instead of 24 hours maybe 8-12 hours would have shown more consistent movement.  Our trials only lasted 5 minutes, increasing that to 20-30 minutes would show more data and hopefully eliminate outliers by giving the tadpoles more time to “explore”.  More colors could also have been implemented into our experiment, which may have not shown a statistically significant differnce for the preference of one color, but may have shown the avoidance of a color which would be data equally if not more informative in the development and evolution of Xneopus laevis.

References

Park, J., Ryu, J., Choi, T., Bae, Y., Lee, S., Kang, H. J., & Kim, C. (2016, October 31). Innate Color Preference of Zebra fish and its use in Behavioral Analyses. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104883/

Xu, X., Scott-Scheiern, T., Kempker, L., & Simmons, K. (2006, July 24). Active avoidance conditioning in zebrafish (Danio rerio). Retrieved from https://www.sciencedirect.com/science/article/pii/S1074742706000797